Inorganic nitrogen

The influence of inorganic nitrogen on microbial CH4 oxidation is exceedingly complex and not yet fully understood. This is because inorganic nitrogen can act as both nutrient and inhibitor for methanotrophy. The role of nitrogen in acting on CH4 concentration, pH and type of methanotroph depends on its form (NH+, NO- or NO-) and concentration. A further complicating factor is the potential inhibiting effect of Cl-when NH+ is applied as NH4Cl (Gulledge and Schimel, 1998; De Visscher and Van Cleemput, 2003a). A review by Bodelier and Laanbroek (2004) on this inhibiting effect was published recently.

At atmospheric CH4 concentrations, inorganic nitrogen is not likely to be the limiting substrate for CH4 oxidation. Consequently, stimulation of CH4 oxidation by adding inorganic nitrogen is rarely observed under such conditions. Most studies indicate that NH4+ and NO2- inhibit CH4 oxidation at atmospheric concentrations, whereas NO3- has no influence other than salt effects (Boeckx and Van Cleemput, 1996; Hutsch et al., 1996; Hutsch, 1998). However, some recent studies indicate that NO3- can be more inhibitive than NH4+ in very acidic soils (Wang and Ineson, 2003; Reay and Nedwell, 2004).

At high CH4 concentrations observed in landfill cover soils, inorganic nitrogen can both stimulate and inhibit CH4 oxidation. These inhibition mechanisms are the same as with atmospheric CH4 concentrations: competitive inhibition by NH4+ (or rather NH3; Carlsen et al., 1991) on the enzyme, toxic effects of NO2- or salt effects (see also Dunfield, Chapter 10, this volume). Stimulation of CH4 oxidation by inorganic nitrogen can be interpreted as a relief of nitrogen limitation of the methanotrophs (De Visscher et al., 1999; Bodelier and Laanbroek, 2004).

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Fig. 12.4. Model fit to the data on the influence of moisture and temperature on methane (CH4) oxidation. (From De Visscher and Van Cleemput, 2000.)

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Fig. 12.4. Model fit to the data on the influence of moisture and temperature on methane (CH4) oxidation. (From De Visscher and Van Cleemput, 2000.)

The effect of inorganic nitrogen on methanotrophy depends on exposure time to CH4 and is closely linked to the micro-bial ecology of CH4 oxidation (De Visscher and Van Cleemput, 2003a; Wilshusen et al., 2004a). As explained in Section 2.1, type I methanotrophs usually grow faster than type II methanotrophs, but the former need a source of inorganic nitrogen, whereas the latter are able to fix N2.

De Visscher et al. (1999) investigated the effect of adding wheat straw and sugar-beet leaves on CH4 oxidation in microcosms imitating landfill cover soils. Adding sug-arbeet leaves, with a low carbon/nitrogen ratio, leads to a net nitrogen mineralization, and this extra inorganic nitrogen source is used by type I methanotrophs for cell synthesis. The result is a stimulation of CH4 oxidation that disappears after 1 month, when the mineralization effect subsides. Adding wheat straw, with a high carbon/ nitrogen ratio, leads to immobilization of the available inorganic nitrogen and immediate nitrogen stress of the type I methano-trophs, allowing the type II methanotrophs to gain an immediate advantage as N2-fixing bacteria. The result is a lasting stimulation of CH4 oxidation.

De Visscher and Van Cleemput (2003a) investigated the influence of exposure time to CH4 on the immediate effect of adding NH4Cl and (NH4)2SO4 to a soil. From this experiment they concluded that soil exposed to high (>1%) CH4 mixing ratios develops methanotrophic activity in three stages. The first stage is a rapid growth of methanotrophs, probably of type I. The second stage is a decline of the methano-trophic activity, probably caused by nitrogen limitation of type I methanotrophs. After a few weeks of steady-state behaviour, a new growth phase - the third stage - is observed, probably dominated by N2-fixing type II methanotrophs. Wilshusen et al. (2004a) came to similar conclusions based on phospholipid fatty acid analysis. They also found that type I/type II selective pressures are related to oxygen availability and extracellular polysaccharide (EPS or exo-polymer) production, as outlined in the following section.

Exopolymer formation

After long periods of exposure to high CH4 mixing ratios, methanotrophic activity sometimes shows a decline that cannot be attributed simply to inorganic nitrogen limitation. It has been suggested that this decline is the result of the production of EPS, which limit the availability of oxygen and CH4 by acting as a barrier to diffusion (Hilger et al., 1999, 2000a; Chiemchaisri et al., 2001; Wilshusen et al., 2004a,b). Figure 12.5 shows a photo of EPS deposits in soils (Hilger et al., 2000a). Although serious detrimental effects of EPS on CH4 oxidation have never been reported on a field scale, this factor is a cause for concern in the development of CH4 biofilters and other engineered controls of microbial CH4 oxidation. It is also becoming increasingly clear that a better insight into ecological controls of EPS production will lead to a better understanding of the ecology of CH4 oxidation itself.

The propensity of methanotrophs to produce EPS was established by Huq et al. (1978). Wrangstadh et al. (1986) hypothesized that oxygen or inorganic nitrogen deprivation leads to the formation of EPS in methanotrophic systems. Wilshusen et al. (2004a) hypothesized that the key to understanding EPS production is N2 fixation by the nitrogenase enzyme. It is well known that nitrogenase activity requires a microaerophilic environment. Whittenbury and Dalton (1981), for instance, found that oxygen concentrations below 4% were necessary to obtain N2 fixation in pure cultures of methanotrophs. Wilshusen et al. (2004a) hypothesized that nitrogen-starved methanotrophs produce EPS as a nitrogen-free carbon sink, allowing them to cycle carbon without the need for inorganic nitrogen. The ensuing pore clogging caused by the EPS creates the microaerophilic environment needed to initiate nitrogenase activity. When the environment is micro-aerophilic from the beginning, nitrogenase activity is initiated without excessive EPS production.

The link between nitrogen limitation and oxygen limitation described here also explains why methanotrophs are known

Fig. 12.5. Soils sampled from columns sparged with synthetic landfill gas. The regions stained with blue dye denote the presence of polysaccharide: (a) dense polymer coated on the soil particles; (b) polymer strands separated from soil particles. (Photo from Hilger et al., 2000a.)

Fig. 12.5. Soils sampled from columns sparged with synthetic landfill gas. The regions stained with blue dye denote the presence of polysaccharide: (a) dense polymer coated on the soil particles; (b) polymer strands separated from soil particles. (Photo from Hilger et al., 2000a.)

as microaerophilic bacteria (Stein and Hettiaratchi, 2001), in spite of the fact that kinetic studies showed a barely perceptible microaerophilic effect (Joergensen, 1985; Bender and Conrad, 1994). Microaerophilic N2 fixation also helps to explain why nitrogen-fixing type II methanotrophs are more competitive at low oxygen concentrations (<30% of saturation in aqueous solution) than at high concentrations. Amaral and Knowles (1995) studied competitive pressure of methanotrophs in gel-stabilized systems and found type II methanotrophs at high CH4/O2 ratios, and type I methanotrophs at low CH4/O2 ratios. Ren et al. (1997), on the other hand, found no evidence of micro-aerophily in pure cultures when CH4 and inorganic nitrogen were not limiting.

Other factors

Arif et al. (1996) found that 5 mg/kg soil dry weight of 2,4-dichlorophenoxyacetic acid (2,4-D) caused partial inhibition of CH4 oxidation by soil. Adding 50 mg/kg soil dry weight caused complete inhibition. Top et al. (1999) used this effect as a biomarker for assessing 2,4-D biodegradation by plasmid-mediated bioaugmentation. Addition of nitrapyrin, a nitrification inhibitor, also caused a strong inhibition of CH4 oxidation (Arif et al., 1996). Boeckx et al. (1998) investigated the influence of various pesticides on CH4 oxidation by arable soils. They found a general decrease of the CH4 oxidation rate. The decrease was statistically significant for lenacil, mikado and oxadixyl in a sandy soil, as well as for mikado, atrazine and dimethenamid in a clayey soil. They also found that landfill cover soils are at least ten times less sensitive to pesticides than arable soils that are not exposed to high CH4 mixing ratios. Borjesson (2001) found that methanethiol and carbon disulphide inhibit CH4 oxidation in landfill cover soils.

Hilger et al. (2000b) found that vegetation (grass) mitigated the inhibiting effect of NH+ on CH4 oxidation in landfill cover soils, probably due to assimilation of the NH+ by the grass. They also found that liming has a positive effect on CH4 oxidation, as the oxidation of 1 mol CH4 generates 0.1-0.12 mol H+.

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